In basic research for hepatitis C virus (hereinafter, also referred to as HCV) and development of anti-HCV drugs, an experimental system that can efficiently amplify HCV is essential. Specifically, a system for amplifying HCV in cultured cells and a system for evaluating the propagation of HCV in cultured cells are necessary, and it is thought that construction of these systems can dramatically advance the above-mentioned research.
HCV is a virus belonging to the flavivirus family of which genome is a single-stranded (+) sense RNA and is known to cause hepatitis C. Recently, it has been revealed that the HCV is classified into many types depending on genotypes or serotypes. According to phylogenetic analysis by Simmonds et al. using nucleotide sequences of HCV strains, it has been found that HCV genotypes are classified into six types, and each type is further classified into several subtypes (Non Patent Literature 1). At present, the full-length genome nucleotide sequences of a plurality of HCV genotypes have also been determined (Non Patent Literatures 2 to 5).
Until recently, infection of cultured cells with HCV and replication of the HCV genome in cultured cells have been impossible. Accordingly, studies on mechanisms of HCV replication and infection have required in vivo experiments using chimpanzees as an experimental animal. However, subgenomic replicon RNAs have been produced from a Con1 strain (GenBank Accession No. AJ238799), an HCV-N strain (GenBank Accession No. AF139594), and an HCV-O strain (GenBank Accession No. AB191333) belonging to the HCV of genotype 1b, and the H77c strain (GenBank Accession No. AF011751), which is the HCV of genotype 1a, and, thereby, studies on HCV replication mechanism can be performed by in vitro experiments using cultured cells (Patent Literature 1 and Non Patent Literatures 6 to 9). Herein, HCV subgenomic replicon RNA means an RNA comprising a part of an HCV genome, which does not have an ability to produce infectious HCV particles, but can autonomously replicate the RNA derived from the HCV genome in cultured cells transfected with it.
In addition, subgenomic replicon RNAs, and fullgenomic replicon RNAs producing infectious HCV particles in in vitro have been produced from a JFH-1 strain, which is an HCV strain of genotype 2a. and, thereby, studies on HCV infection mechanism can also be performed by in vitro experiments using cultured cells (Non Patent Literatures 10 and 11). Herein, the fullgenomic replicon RNA of HCV means an RNA comprising a full-length HCV genome, i.e., an RNA comprising a 5′ UTR, structural genes, non-structural genes, and a 3′UTR. and can autonomously replicate the RNA derived from the HCV genome in cultured cells transfected with it.
Pietschmann et al. have investigated production of infectious HCV particles using HCV fullgenomic replicon RNAs of a Con1 strain of genotype 1b and of a mutant having replication enhancing mutation. The replication level of the viral genome is, however, significantly low, and the current situation is therefore that detection of the HCV particles of genotype 1b requires addition of a casein kinase 1 inhibitor enhancing replication of the replicon RNA of HCV to a cell culture system or an infection experiment by an in vivo system using an animal (Non Patent Literature 12). This means that, at present, only RNAs derived from the JFH-1 strain of genotype 2a can produce infectious HCV particles in an in vitro system using cultured cells and that only HCV JFH-1 strain or fullgenomic replicon derived from the JFH-1 strain can produce a large amount of HCV particles in an in vitro system for obtaining an HCV vaccine raw material.
Incidentally, current main therapy for hepatitis C is monotherapy with interferon-α or interferon-β and combined therapy with interferon-α and ribavirin, a purine nucleoside derivative. The therapeutic effect of such therapy, however, is recognized in only about 60% of the total subjects, and it is known that even in patients to whom the therapy was effective, hepatitis C recurs in more than the half thereof by stopping the therapy. The therapeutic effect of interferon is associated with HCV genotypes, and it is known that the effect on HCV of genotype 1b is low and that the effect on HCV of genotype 2a is higher (Non Patent Literature 13). The causes why the therapeutic effect of interferon varies depending on HCV genotypes are still unknown, but a difference in replication mechanism or replication efficiency of HCV is thought to be one of the causes.